Document Type

RU Laboratory

Keywords

Abstract

The regulation of apoptosis, or programmed cell death has been the subject of a vast body of research because of its implication in normal development, tissue homeostasis and a wide range of diseases. The major point of focus for understanding apoptosis regulation is the activation of its primary executioner: the caspase. This family of proteolytic enzymes has been shown to be tightly controlled, as many different proteins govern their transcription, stability, activation, and activity. Consequently, caspase regulation is extremely complex, and is further complicated by a discrepancy between different cell types and paradigms. Caspases have also been shown to have non-apoptotic roles in many vital cellular processes, heightening the need for specific modulators. Therefore, exactly how caspases are regulated is still being worked out, and new factors are still being discovered. In this thesis, I describe the isolation of new caspase regulators. From a genetic screen for mutants that abrogate caspase activity during terminal spermatid differentiation, we isolated several mutants that have unique roles in caspase activation. The screen revealed 33 mutants that complement into 22 genetic groups. Although they all share a deficiency in caspase activation, these mutants were further sub-divided into groups with distinct morphological phenotypes. One group mapped to cytochrome-c-d, previously shown to be required for caspase activation in this system. We also isolated two members of a Cullin-3-based ubiquitin ligase complex, Cullin-3T and KLHL10. The mutations in cullin-3 were in a specialized intron that splices into a testis-specific isoform, the first characterization of a tissue-specific isoform of this gene. It was the first report describing the function of this type of ubiquitin ligase in caspase regulation. Another isolated mutant was in a yet uncharacterized F-box protein we termed Nutcracker. F-box proteins are the substrate-binding units of SCF ubiquitin ligases, and we showed that Nutcracker is indeed part of such a complex. From a proteomics screen for Nutcracker physical interactors, we isolated DmPI31. Nutcracker promotes DmPI31 stability, and a complex between these proteins is essential for both caspase activation and sperm differentiation. DmPI31 is conserved with the mammalian PI31 proteins, initially described as proteasome inhibitors. By contrast, we show that DmPI31 is a proteasome activator both in vitro and in vivo. Generation of dmPI31 mutants by homologous recombination revealed that dmPI31 is required for viability and fertility, as the targeted depletion of dmPI31 resulted in sterility due to meiotic cell-cycle abnormalities. These results define a new mechanism for the regulation of proteasome function. The overall findings demonstrate that cellular remodeling during spermatid differentiation requires the coordinate action of two major proteolytic entities: caspase and ubiquitin-proteasome systems.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.